Fig 1: The images of H.E. staining and immunohistochemistry staining for prenatal rat dental germs. H.E. staining demonstrated that the rat dental germs entered the early cap stage at E14.5 d, the cap stage and early bell stage at E16.5 d, and the bell stage at E18.5 d. The separation between the pre-odontoblast and pre-ameloblast layers occurred in all E18.5 d species. Immunohistochemistry staining revealed that Clock, Per1, and Col1 were detected in the epithelial-mesenchymal interaction area, dental follicle, and dental papilla at E14.5 d, and became stronger at E16.5 d when p75NTR, Bmal1, and ALP were detected. All the factors were expressed at E18.5 d, but Cry1 showed the weakest expression. All experiments were repeated three times independently. op: oral epithelium; dp: dental papilla; iee: inner enamel epithelium; oee: outer enamel epithelium; sr: stellate reticulum. The scale bar represents 50 µm, respectively.
Fig 2: The in vitro observation of circadian rhythm dynamics in rat E18.5 d EMSCs. Quantitative RT-PCR analyses depicted that p75NTR mRNA expression patterns were similar in three conditions: higher at about ZT4, ZT24, and ZT36-44 and lower at about ZT16, ZT32, and ZT48. However, the patterns had highest amplitudes, and a supramaximal peak was detected at ZT4 in the D.D. condition. Of the three clock genes, Bmal1 and Clock showed a similar mRNA expression pattern to p75NTR in three conditions. Per1 was similar in the L.L. condition, but four, not three, peak times presented in the L.D. and D.D. conditions. The mineralization-related factors Runx2 also showed a similar mRNA expression pattern to p75NTR in L.L. and D.D. conditions, but four peak times were observed in the L.D. condition: ZT0, ZT24, ZT32, and ZT44. The odontogenesis-related factor Dlx1 showed irregular oscillating expressions: ZT4, ZT20, ZT32, and ZT40 in the L.L. condition; ZT8, ZT16, ZT28, ZT40, and ZT40 in the D.D. condition; and ZT0, ZT24, and ZT44 in the L.D. condition. Besides, the peak at ZT28 in all the six detected factors in D.D. condition, but it was replaced by a V-shape (as indicated by the red arrows) in the L.D. condition when the cells were exposed to the second 12-h light stimulus. All experiments were repeated three times independently. Data are expressed as mean ± S.D. n = 13 per group.
Fig 3: Effect of PCL immobilized NRG1 on KC-NC differentiation and proliferation toward towards Schwann cells. Immunofluorescence images of the expression of Schwann marker, S100b, GFAP, p75, and myelin PLP1 on (A–C) tissue culture plates (TCP), (D–F) electrospun fibers with no coating (Control), or (G–I) immobilized NRG1. (A–C) scale bars: 50 µm; (D–I) scale bars: 20 µm. (J) Quantification of the mean immunofluorescence intensity (MFI) of the indicated SC markers normalized per cell on TCP, control fibers, and NRG1 modified fibers. n = 50 randomly selected cells from different fields. (K) Quantification of average cell density at two-time points (3 DIV and & DIV, DIV = Day in-vitro) on TCP, control, or NRG1 modified fibers. Data are presented as Mean ± SD, One-way analysis of variance (ANOVA) with Tukey post hoc test. ns: p = 0.05), ****: p <0.0001.
Fig 4: The in vivo assays for the effect of p75NTR knockout on the mRNA expression in dental germs at PN7d. As expected, p75NTR mRNA in the knockout mice was significantly lower than in THE wild-type mice (p < 0.05). Mage-D1, Bmal1, ALP, Col1, Msx1, Dmp1, and Dspp showed the same change in the p75NTR knockout mice, indicating a positive relationship with p75NTR. In contrast, Runx2 showed the reverse change, presenting a negative relationship with p75NTR. Clock, Per1, Per2, and Dlx1 showed no significant difference between p75NTR knockout and wild-type mice (p > 0.05). All experiments were repeated at least three times independently. Data are expressed as mean ± S.D. L. D. Condition group, n = 6; D. D. Condition group, n = 6.
Fig 5: The in vitro assays for the effect of p75NTR over-expression on the mRNA expression in immortalizing stem cells from dental apical papilla (iSCAP). p75NTR was expected to be significantly higher expressed in the over-expression group than that in the control group (p < 0.05). Mage-D1, Bmal1, Clock, Runx2, Col1, and Msx1 showed an apparent positive relationship when p75NTR was significantly up-regulated. In contrast, ALP, Dmp1, and Dspp showed a negative relationship with p75NTR. Per1, Per2, ALP, Dlx1, and Dmp1 showed no significant difference between the over-expression group and control group (p > 0.05) with lower than that in wild-type mice (p < 0.05). All experiments were repeated at least three times independently. Data are expressed as mean ± S.D. Overexpression negative control group, n = 3; p75NTR over-expression group, n = 3.
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